In fluorescence imaging, there is often a need for detecting the presence of multiple fluorescent labels (fluorophores) in a given sample simultaneously. Various methods may be employed to discriminate fluorescent labels.
In a first method, light emitted by the labels is split over multiple detectors according to wavelength, for instance, by using dichroic mirrors.
Another method uses the fact that different labels are usually sensitive to different wavelengths of light for excitation. A set of light sources, e.g. lasers, differing in their respective wavelengths, is operated in an alternating manner. Each of the light sources typically excites a particular set of fluorescent labels. Separate images of the various labels can be obtained by reading out each detector before switching to the next light source, i.e. before changing the frequency of the illumination light. The technique is an example of time sharing or time domain multiplexing. An advantage is that it allows using one detector for detecting light of different frequencies. The technique thus avoids the need for providing a separate detector for each wavelength.
The two methods can be combined so as to increase the number of fluorophores that are detectable using a fixed number of light sources. It is noted that the total number of fluorophores that can be detected independently may be larger than both the number of different light sources and the number of detectors.
In a scanning microscope with time domain multiplexing, the total integration time per scan is increased as compared to detecting a single type of fluorophore per sensor. However, time domain multiplexing is still advantageous over doing multiple scans of the same area for at least two reasons. Firstly, less time is lost to ‘overhead’ for scanning the sample (for, e.g., reversing the scan direction or moving the sample back to a start position for the next scan). Secondly, changes in the system over time have less influence on a mutual alignment of images obtained from different excitation and/or fluorescence wavelength.
Time domain multiplexing is suitable, among others, for confocal scanning (using, for example, non-pixelated sensors), simple line sensors and full frame sensors. In the case of a line sensor and a continuously moving sample, the light sources are usually switched every time the pixel rows have been read out, so that after a single scan two or more different full images of the same object have been acquired.
Time delay and integration (TDI) is an imaging method known to be often faster compared to using a simple line sensor. It can be applied both to brightfield and other imaging modalities. TDI typically uses a special charge coupled device (CCD) having multiple adjacent rows of pixels. As an image of the object is scanned continuously over the sensor, the accumulated charge on the sensor is moved in a synchronous manner from each row of pixels to the next row. Each time, only a signal from the last row of pixels is read out and stored in memory. In this way a signal is accumulated on the sensor with a much longer integration time than is possible on a simple line sensor at the same scan speed.
In summary, for various applications of scanning fluorescence imaging it is often considered advantageous to detect multiple fluorophores on an object under study simultaneously. To this end, multiple light sources emitting light at different wavelengths can be employed. The light sources can be operated so as to expose the object to light at different wavelengths in an alternating fashion. A sensor detecting fluorescent light from the object may be read out between two consecutive illumination periods.
FIG. 1 illustrates the principle of TDI imaging. An object 12 is illuminated by a light source (not shown) and moved with constant speed along an object path 14. In the Figure, only an exemplary light-emitting point of the object 12 is graphically represented. Imaging optics 20 comprising, for example, a lens or a lens system, generates an optical image 24 of the object 12 on a TDI sensor 22. As the object 12 moves along the object path 12, its image 24 moves across the TDI sensor 22 along an image path 26. In the example shown, the object path 14 is a straight line, but other paths may be envisaged, depending on the design of the TDI sensor 22. In the example shown, the image path 26 is also a straight line. Note that the object 12 and its image 24 move in opposite directions, as indicated by the arrows 14, 26. The sensor 22 comprises a plurality of parallel rows 28, each row comprising a plurality of pixels (cells). The rows 22 comprise a first row 30 and a last row 32. While the object 12 is located at an initial position as shown in the Figure, light 16 emitted by the object 12 is incident on the first row 30. As the object 12 is at a final position (corresponding to the tip of the arrow 14), light 18 emitted by the object is incident on the last row 32 of the sensor 22. Charge is accumulated on the pixels of the TDI sensor 22 as a function of both the intensity of the optical image 24 and the time during which the pixels are exposed to the image 24. The accumulated charge is shifted through the sensor 22 synchronously with the movement of the optical image 24. The signal built up by the sensor 22 is therefore larger than that of an equivalent simple line sensor by a factor that equals the number of pixel rows 22. During two consecutive readouts of the TDI sensor 22 the object 12 moves over a scan length that can be quite considerable in comparison to the size of the features of interest of the object 12. A problem, however, is that switching between imaging modes, such as switching of light sources, before reading out the entire TDI sensor 22 would result in mixing of the corresponding images on the sensor 22.
It is an object of the invention to provide a time domain multiplexing TDI imaging method. It is another object of the invention to provide a time domain multiplexing TDI imaging system. It is yet another object of the invention to provide a TDI sensor for being used in a time domain multiplexing TDI imaging system.
These objects are achieved by the features of the independent claims. Further specifications and preferred embodiments are outlined in the dependent claims.